Actin plays a central role in cell motility. As cells move in response to external signals, the cytoskeleton is constantly remodeled. For this process to continue, actin subunits must be continuously recycled to the leading edge. Although the assembly rates of actin in vitro are extremely rapid, the pointed end disassembly rate constant measured in vitro is far too slow to recycle the monomers necessary for this process to continue. Recent studies have identified a family of actin binding proteins that play a central role in actin filament dynamics in cells by both severing filaments and increasing the rate of disassembly from ends. Cofilin and ADF (Actin Depolymerizing Factor) are members of a large family of proteins that is conserved across the complete spectrum of eukaryotic organisms. They are key players in actin filament turnover in yeast and are essential in all organisms tested. Failure to regulate cofilin properly leads to Williams syndrome in man, while point mutations in cofilin produce paralysis in Caenorhabiditis elegans. Thus, elucidating how cofilin regulates actin assembly has important implications for understanding both normal and diseased states in cells. In electron cryomicroscopy studies published last year, I showed that cofilin dramatically alters F-actin structure when it binds. The purpose of the proposed research is to test the hypothesis that this effect on F-actin structure represents a novel mechanism for regulating actin dynamics and assembly in cells. The first goal of this study is to model cofilin/F-actin interactions in greater detail. Cofilin will be labeled with gold on specific residues which will be used as markers to accurately position the x-ray model in our reconstruction. In addition, we will extend the resolution of the current reconstruction beyond its present limit (27 Angstrom units) using improved experimental and computational methods. The second goal of this proposal is to use both genetically-engineered and naturally-occurring variants to identify the specific residues that endow cofilin with its unique actin regulatory properties. The third goal is to explore the molecular basis of a cofilin-dependent muscle disease in C. elegans by determining how cofilin mutants responsible for this disease interact with F-actin. This project will test the hypothesis that cofilin's effects on actin filament structure are central to its function in multi-cellular organisms. Our fourth goal is to explore the hypothesis that cofilin promotes the formation of alternative actin assemblies in cells through its mode of binding to the filament. This will involve structural studies of non-helical cofilin/actin assemblies produced in vitro as well as of cofilin/actin rods found in the cytoplasm and nuclei of diseased cells.